Procedure for obtaining carbocalcitonin

ABSTRACT

A procedure for obtaining carbocalcitonin comprising the condensation of fragment 1, a docosapeptide corresponding to the carboxamide end of the carbocalcitonin sequence, conveniently protected and anchored on resin, with fragment 2 or 3, a nonapeptide corresponding to the amino end of the salmon calcitonin sequence, with a ready-formed cycle between the Asu and Ser residua, and the treatment of the complete peptide skeleton (fragment 6 or 7) with an acid to free the totally deprotected peptide from the resin. ##STR1##

The present invention relates to a procedure for solid phase preparationof salmon and eel carbocalcitonin and all of its pharmaceuticallyacceptable salts formed by acid addition or complexes thereof. Theinvention further relates to the preparation of intermediate compoundswhich are useful in the synthesis of carbocalcitonin according to theinvention, in particular the preparation of two aminosuberic acidderivatives, not previously described, the w-allyl ester of aminosubericacid and the w-allyl ester ofN-α-9-fluorenylmethoxycarbonyl-aminosuberic acid.

BACKGROUND OF THE INVENTION

The carbocalcitonins are unnatural peptides which are analogous to thecalcitonins but which are very much more active. Like the calcitoninscarbocalcitonins have important therapeutic applications for thetreatment of hypercalcemia, osteoporosis or Paget's disease by virtue ofits ability to regulate the level of calcium in the blood. Thesecompounds have greater stability in serum, the liver or the kidneys thannatural calcitonins and are more stable during the process ofpurification and in storage due to the absence of the disulphide bridgewhich is present in the calcitonins.

Naturally occurring calcitonins, such as those of eel, salmon or humans,are polypeptides of 32 amino acids, the residue 1 and 7 beingL-cysteine, whose side chains are linked to form a disulphide bridge.Carbocalcitonins do not have L-cysteine in said positions, position 7being occupied instead by L-α-aminosuberic acid (Asu) with the formula:##STR2## the w-carboxyl group of which is linked to the terminal aminogroup of the peptide to produce a cyclic structure.

In order to generate the cycle between the Asu side chain and theterminal amino end in a selective way which avoids secondary reactions,the Asu side chain must be protected with a group which is orthogonal tothe rest and to the peptide-resin link. The present invention describesa procedure for obtaining the new aminosuberic acid derivative with the,side chain protected with the allyl group and the α-amino protected bymeans of Fmoc. ##STR3## This protective group is eliminated by means ofpalladium catalysis and is totally orthogonal to the other protectivegroups and the peptide-resin link (Greene T. W. Protective Group inOrganic Synthesis, Wiley, N.Y., 1981, p.169).

BRIEF DESCRIPTION OF THE TABLES

Tables 1-4 diagram the procedure for obtaining a carbocalcitonin basedon solid phase synthesis according to the process of the presentinvention. The "Fragments" identified in the Tables correspond to theamino acid sequences as follows:

(Fragment 1=SEQ. ID NO: 1; Fragments 2 and 3=SEQ. ID NO: 4; Fragments 4and 5=SEQ. ID NO: 3; Fragments 6 and 7=SEQ ID NO: 5; Fragments 9 and10=SEQ. ID NO: 2; and Carbocalcitonin=SEQ. ID NO: 6.)

If, however, a discrepancy were to exist between the fragments shown inthe tables and specification and the Sequence Listing, due totypographical error, the fragments shown in the tables and specificationare deemed to constitute the invention.

Furthermore the invention describes a procedure (Table 1) for obtainingcarbocalcitonin using said Asu derivative, in particular for obtainingthe analogues of salmon and eel calcitonin, the formula (SEQ. ID NO: 6)of which is expressed as follows: ##STR4## where AA₂₅ is Asp or Asn,AA₂₆ is Val or Thr and AA₂₈ is Ala or Ser.

More particularly, the present invention provides a procedure forobtaining carbocalcitonin based on the solid phase synthesis usingFmoc/tBu type protective group methodology (with convenientfunctionalized supports) combined with a convergent strategy, allaccording to the following (Table 1):

    TABLE 1                                                                          -                                                                              ##STR5##                                                                       ##STR6##                                                                       ##STR7##                                                                       ##STR8##                                                                      ##STR9##                                                                       As shown, ® refers to the ® of Table 1, namely the solid support,      internal standard and bridge group having the structure shown after the        colon.                                                                    

Referring to FIG. 1 above, the procedure of this invention consists ofthe condensation of fragment 1 (SEQ ID NO:1), a docosapeptidecorresponding to the carboxamide end of the carbocalcitonin sequence,conveniently protected and anchored on resin, with fragment 2 (SEQ. IDNO:4), a nonapeptide corresponding to the amino end of thecarbocalcitonin sequence, with the cycle already formed between the Asu⁹and Ser¹ residues, to obtain fragment 6 (SEQ. ID NO:5). Another optionconsists of coupling fragment 3 (SEQ. ID NO:4), which is also cyclic, tofragment 1 (SEQ. ID NO:1), thereby obtaining fragment 7 (SEQ. ID NO:5),all according to what is shown in Table 1.

Once the complete peptide skeleton (fragment 6 or 7) (SEQ. ID NO:5) hasbeen constructed the peptide is liberated from the resin and totallydeprotected, already in its cyclic form, by means of an acid treatment.Finally, after subsequent purification, chemically pure carbocalcitoninis obtained.

In particular, the condensation of fragment 1 (SEQ. ID NO:1) withfragment 2 or 3 (SEQ. ID NO:4) (Table 1) which leads to fragment 6 or 7(SEQ. ID NO:5) is carried out by the conventional methods of solid phasesynthesis which have already been described. Once condensation iscomplete, the peptide-resin is subjected to a simultaneous process ofdeprotection of the side chains and de-anchoring the resin usingtrifluoroacetic acid in the presence of carbocation sequestering agents.The resulting crude is purified by high pressure liquid chromatographyand ion exchange chromatography. The collection of homogeneous fractionsis combined and freeze-dried, dried, thereby obtaining carbocalcitoninIn the free state.

According to another modality, the procedure of the invention may becarried out according to the following Table 2:

    TABLE 2                                                                          -                                                                              ##STR10##                                                                      FmocSer(X)AsnLeuSer(X)Thr(X)Asu(OAI)ValLeuGlyLys(Boc)LeuSer(tBu)GlnGlu(O      tBu)LeuHis(Trt)Lys(Boc)                                                         ##STR11##                                                                      ##STR12##                                                                      ##STR13##                                                                      ##STR14##                                                                

where R is defined as before.

According to Table 2, the cycling can also be carried out aftercondensation. To do this there two possibilities: condense fragment 1SEQ. ID NO:1, corresponding to the docosapeptide, with fragment 4 (SEQ.ID NO:3) to obtain fragment 9 (SEQ. ID NO:2), or condense fragment 5(SEQ. ID NO:3) to obtain 10 (SEQ. ID NO:2), as shown in Table 2.Alternatively, fragment 10 (SEQ. ID NO:2) can be obtained by means oflinear synthesis, i.e. by incorporating one amino acid residuum afteranother until sequence is complete, as shown is Table 3.

Once the skeleton corresponding to fragments 9 or 10 (SEQ. ID NO:2) hasbeen obtained the deprotection of the terminal amino of the resin(elimination of the Fmoc group) and the side chain of the Asu residuum(elimination of the allyl group) is carried out to obtain fragments 6 or7 (SEQ. ID NO:5). Afterwards, and in the same way for both cases, thepeptide-resin is treated with trifluoroacetic acid in the presence ofcarbocation captivators to deprotect and liberate the peptide from theresin. After purification, carbocalcitonin in its free state isobtained.

According, to another aspect and as mentioned above, the inventionprovides a procedure for obtaining said fragment 1 (SEQ. ID NO:1), aswell for obtaining fragment 5 (SEQ. ID NO:3), the peptide-resin which isthe precursor of fragment 3 (SEQ. ID NO:4), and for fragment 4 (SEQ. IDNO:3), the peptide-resin which is the precursor of fragment 2 (SEQ. IDNO:4) all of them used an Table 1 and 2 above for the preparation ofcarbocalcitonin according to the present invention.

Fragments 1 (SEQ. ID NO:1), 4 and 5 (SEQ. ID NO:3) are obtained startingwith paramethylbenzhydrilamine resin (PMBHA) and incorporating aninternal standard and a bridge group (handle) between the resin and theamino acids of the respective sequences.

To obtain fragment 1 (SEQ. ID NO:1), and because the terminal carboxyend of carbocalcitonin is a carboxyamide, the bridge group or handlewhich is incorporated is 1-(9H-fluoren-9-yl) methoxy-formamide!methyl-3,5-dimethoxyphenoxyvaleric acid (Fmoc-PAL) (Albericio et al. J.Org.Chem. (1990), 55, 3730) or alternatively p- (R,S)-α- 1-(9H-fluoren-9-yl)methoxy-formamide!-2,4-dimethoxybenzyl!-phenoxy acetic acid (Fmoc-AM)(Atherton et al. J.Am. Chem. Soc. (1975), 97,6584. The incorporation ofthe bridge group is followed by linear synthesis, residuum by residuum,until the 22 amino acids are incorporated, using Fmoc as the protectivegroup for the amino end in all cases. For the side chains Lys, Ser, Thr,Tyr Arg, Glu, His, Gln, Asn and Asp the protective groups are thoseindicated in the following Table 3:

                                      TABLE 3                                     __________________________________________________________________________     ##STR15##                                                                     ##STR16##                                                                     ##STR17##                                                                    __________________________________________________________________________

where R is defined as before.

To obtain fragment 4 (SEQ. ID NO:3), and because the terminal carboxyend must be carboxyacid, the resin 4-(hydroxymethyl)phenoxymethyl-copoly(styrene-1%divinylbenzene! (Wang resin) (Lit. S.:Wang.JACS1973,95,1328).

To obtain fragment 5 (SEQ. ID NO:3) and again because the terminalcarboxy end must be carboxyacid, the bridge group or handle incorporatedis 4(4-hydroxymethyl-3-methoxy-phenoxy)butyric acid (HMPB), also knownas the Riniker Handle (Florsheimer et al.). In both cases theincorporation of the bridge group is followed by linear synthesis,residuum by residuum, until the nine amino acids are incorporated, usingthe Fmoc group as the protective group for the amino end in all casesand the tBu group for the side chains Ser and Thr, as indicated in thefollowing Table 4:

    TABLE 4                                                                          -                                                                              ##STR18##                                                                      ##STR19##                                                                      ##STR20##                                                                

According to the previous table and to tables 1 and 2, the cyclizationof the nonapeptide can be carried out before or after it has beenincorporated with the fragement of 22 amino acids (fragment 1). To dothis the side chain of the Asu residuum must be deprotected, this beingdone by means of Pd catalysis as has already been mentioned.Furthermore, the terminal amino end must also be deprotected,eliminating the Fmoc group with piperidine/DMF. The cycling is carriedout by means of the standard method of forming amide bond, in particularwith DIPCDI.

Referring once again to Table 4, the same steps as described above canalso be carried out with the nonapeptide partially deprotected, i.e.without the tBu groups (fragments 2 (SEQ. ID NO:3) and 4(SEQ. ID NO:3)),working with Wang resin as shown in the diagram.

The abbreviations used in the present description have the followingmeanings:

AcOH: acetic acid

AcOEt: ethyl acid

Al: allyl

Ala: L-alanine

AM: p- (R,S)-2,4-dimethoxy benzyl!-phenoxy acetic acid

Arg: L-arginine

Asn: L-asparagine

Asp: L-aspartic acid

Asu: L-α-aminosuberic acid

Boc: t-butoxycarbonyl

DCM: dichloromethane

DIEA: N,N'-diisopropyllethyl amine

DIPCDI: dilosopropylcarbodiimide

DMAP: dimethylaminopiridine

DMF: N,N'-dimethylformamide

EDT: 1,2-ethandithiol

Fmoc: 9-fluorenylmethoxycarbonyl

Fmoc OSu: 9-fluorenylmethoxycarbonyl N-hydroxysuccinimide ester

Gln: L-glutamine

Glu: L-glutamic acid

Gly: L-glycine

HBTU: 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniohexafluorophosphate

His: L-histidine

HMPB: 4(4-hydroxymethyl-3-methoxyphenoxy)butyric acid

HOBT: N-hydroxybenzotriazole

HPLC: high pressure liquid chromatography

Ile: L-isoleucine

Leu: L-leucine

Lys: L-lysine

MeOH: methanol

PAL: aminomethyl-3,5-dimethoxyphenoxyvaleric acid

pMBHA: para-methylbenzhydrilamine

Pmc: 2,2,5,7,8-pentamethylchroman-6-sulphonyl

Pro: L-proline

PyBOP: benzotriazole-1-yl-oxy-tris-pirrodoline-phosphonidehexafluoraphosphate

Ser: L-serine

tBu: tert-butyl

TFA: trifluoroacetic acid

THF: tetrahydrofuran

Thr: L-treonine

TLC: thin film chromatography

Tos: tosyl

Trt: trityl

Tyr: L-tyrosine

Val: L-valine

The invention is illustrated below by means of the followingnon-limiting examples, in which AA₂₅ Asp or Asn, AA₂₆ =Val or Thr, AA₂₈=Ala or Ser and AA'₂₅ =Asp(OtBu) or Asn, AA'₂₆ =Val or Thr(tBu), AA'₂₈=Ala or Ser(tBu).

EXAMPLE 1

Protection of side chain of aminosuberic acid (Asu). Obtaining w-allylester of L-α-aminosuberic acid (H-Asu (OAl)--OH).

1.134 g (6 mmol) of L-α-aminosuberic acid are suspended in 30 ml ofallylalcohol (previously dried over a 3 Å sieve. In a nitrogenatmosphere, 1.9 ml (15 mmol) of chlorotrimethylsilane are added to thesuspension drop by drop and the resulting solution is shaken for 20hours at room temperature, the progress of the reaction being checked bymeans of TLC (AcOEt/AcOH 99/1). 200 ml of cold diethyl ether are addedand the precipitate which appears is separated by centrifuging. It isthen resuspended in ether and the same operation is repeated two moretimes. 915 g of a white solid are obtained, corresponding to thehydrochloride of the product. The mother water cools down (+4° C.) andthe precipitate which appears is also centrifuged and washed with etherto obtain 135 mg more the product. The overall yield is 66%.

¹ H RMN (CD₃ OD, 200 MHz)δ: 5.93 (ddt, 1H, --CH═CH₂), 5.4-5,15(m, 2H,--CH═CH₂), 4.58 (d, 2H, J=5.5 Hz, COO--CH₂), 3.97 (t, 1H, J=6 Hz,CH--COOH), 2.33 (t, 2H, J=7.32 Hz, CH₂ --COO), 2.05-1.3 (m, 8H, CH--CH₂--CH₂ --CH₂ --CH₂).

EXAMPLE 2

Protection of α-amino group of aminosuberic acid. ObtainingFmoc-Asu(OA)--OH.

1.05 g (.3.9 mmol) of Asu(OAl)-HCl are dissolved in 25 ml of an aqueoussolution of 10% Na₂ CO₃. ensuring that the pH does not exceed the valueof 10. The resulting solution is cooled to 0° C. and a suspension 1.31 g(3.9 mmol) of Fmoc-OSu in 6.5 ml of acetone is added drop by drop. Thesuspension is shaken constantly and kept at the same temperature for 1hour. When this time has elapsed it is left to reach room temperatureand the reaction is checked with TLC (CHCl₃ /MeOH/AcOH 90/8/2). After 3hours the suspension will have disappeared and a transparent solution isobserved. The solution is poured onto 200 ml of a mixture of ice andwater and washed diethyl ether (4×30ml). The aqueous phase is cooled to0° C. and acidified to pH=5.5 using dilute HCl solution, observing theprecipitation of the Fmoc-ASu(OAl)--OH. The suspension is extracted withAcOEt (4×100 ml), reacidifying in each extraction in order to maintain apH of 5.5. The collection of organic phases is dried on MgSO₄ and thesolvent is eliminated under low pressure. An oil is thereby obtainedwhich crystallizes to give the product in the form a white, friablesolid (1.39 g/yield 79%).

¹ H RMN (CDCl₃, 200 MHz) δ: 8.07 (width), 7.8-7.2 (m, 8H), 5.9 (ddt,-1H, --CH═CH₂), 5.4 (d, 1H, J=9.7 HZ; CH (Fmoc)), 5.35-5,15 (m, 2H,--CH═CH₂), 4.58 (d, 2H, CH₂ --COO--CH₂), 4.43 (dd, 2H, CH₂ (Fmoc)), 4.21(t, 1H, CH--COOH), 2.34 (t, 2H, J=7.6 Hz, CH₂ --COO--), 2-1.2 (m, 8H,CH₂ --CH₂ --CH₂ --CH₂ --COO--).

EXAMPLE 3

Incorporation of an internal standard. Obtaining Boc-Ile-pMBHA.

1.658 g (6.9 mmoles) of BocIle are incorporated onto 4 g ofp-methylbenzhydrilamine resin of 0.69 mmol/g resin as an internalstandard by means of the synthesis programme described below:

    ______________________________________                                        Step      Reagent     Repetitions                                                                             Time                                          ______________________________________                                        1         TFA 40%     1         2'                                            2         TFA 40%     1         20'                                           3         DCM         5         1'                                            4         DIEA 5%     3         2'                                            5         DCM         5         1'                                            6         Boc aa      -         +                                             7         HOBt        -         +                                             8         DIPCDI      -         40'                                           9         DCM         5         1'                                            10        check with ninhydrin, if + return to 6, if - continue               11        DCM         5         1'                                            12        TFA 40%     1         2'                                            13        TFA 40%     1         20'                                           14        DCM         5         1'                                            15        DIEA 5%     3         2'                                            16        DCM         5         1'                                            ______________________________________                                    

EXAMPLE 4

Incorporation of Riniker handle. Obtaining4(4-hydroxy-methyl-3-methoxyphenoxy) butyramide-Ile-pMBHA.

There then follows the incorporation of 4(4-hydroxymethyl-3-methoxyphenoxy) butyric acid, also known as the Riniker handle(HMPB). This is carried out by reacting 4 g of the resin (fNH₂ =0.69.mmol/g of resin), after it has been previously functionalized with theinternal standard, with 1 g (4.14 mmoles of, 1.5 equivalents) of4(4-hydroxymethyl -3-methoxyphenoxy) butyric acid, 0.62 g (4.14 mmoles,1.5 equivalents) of HOBt and 641 μl (4.14 mmoles; 1.5 equivalents) usingDMF as the solvent. The reaction time is 90'. After this time haselapsed, the resin is washed five times with DCM and the Kaiser is usedto check that there are no free amines. If there are, the couplingprocess must be repeated.

EXAMPLE 5

Incorporation of the first amino acid. Obtaining Fmoc-Gly-Rinikerhandle-Ile-pMBHA.

The incorporation of the first amino acid, in this case glycine, impliesthe formation of an ester-type link between the handle and the Fmoc Glyderivative. For this kind of incorporation the resin is reacted with 4.1g (5 equivalents) of Fmoc Gly in the presence of 168 mg (0.5equivalents) of DMAP and 2.094 ml (5 equivalents) of DIPCDI in DMF for90". Once the reaction is complete the resin is washed five times withDMF. An amino acid analysis or an acid hydrolysis of the resin gives theratio of the Ile amino acid (internal standard) and the first amino acidGly. In this way the real functionalization of the resin is known,normally varying between 0.35-0.69 mmol/g.

EXAMPLE 6

Incorporation of the first amino acid onto the Wang resin. ObtainingFmoc-Gly-Wang resin.

This involves the same steps as example 5 except that the amino acidanalysis is not carried out due to the absence of the internal standard.

EXAMPLE 7

Incorporation of the remaining amino acids. Obtaining FmocSer.sup.(tBu)-Asn-Leu-Ser(tBu)-Thr(tBu)-Asu(OAl)-Val-Leu-Gly-handle Riniker-Ile-pMBHAor FmocSer(tBu)-Asn-Leu-Ser(tBu)-Thr (tBu)-Asu(OAl)-Val-Leu-Gly-Wangresin (SEQ. ID NO:4).

The incorporation of the remaining amino acids is carried out byfollowing a synthesis programme such as the one described below:

    ______________________________________                                        Step      Reagent      Repetitions                                                                             Time                                         ______________________________________                                        1         DMF          5         1'                                           2         pip/DMF 20%  1         1'                                           3         pip/DMF 20%  1         5'                                           4         DMF          5         1'                                           5         Fmoc aa      --        +                                            6         HOBt         --        +                                            7         DIPCDI       --        40'                                          8         DMF          5         1'                                           ______________________________________                                    

check with ninhydrin, if + return to 5, if - continue with step 1 andthe next amino acid.

In order to evaluate the synthetic purity of the totally deprotected 1-9peptide, 20 mg of H-Ser(tBu)-Asn-Leu-Ser(tBu)-Thr(tBu)-Asu(OAl)-Val-Leu-Gly-Riniker handle-Ile-pMBHA (SEQ. IDNO:7) are treated with 900 μl of TFA, 50 μl of thioanisole, 30 μl of EDTand 20 μl of anisole for 2 hours at room temperature in a reactorprovided with a filter plate (this treatment does not affect the allylgroup, which remains unalterable). The filtrate is collected in a tubewith cold, dry diethyl ether. The precipitation of the free peptide isobserved and after centrifuging the floating material is decanted. Thepellet is resuspended once again in cold, dry ether to eliminate thescavangers (EDT, thioanisole, anisole). This operation is repeated fivetimes. Afterwards the pellet is dried and then dissolved in 1 ml of 10%acetic acid solution. 40 μl of the peptide solution are injected intoHPLC with a gradient of 5-85% B, where A: H₂ O 0.045% TFA and B: CH₃ CN0.035% TFA, in a Vydac column C18 5 μm, 25×0.46 cm. An amino acidanalysis of a hydrolysis of the peptide-resin at 150° C. for 3 hourswith a mixture of HCl/propanoic acid gives the following composition Asp1.01 (1), Thr 0.8 (1)0 Ser 1.8 (2), Gly 1.2 (1), Ile 1.5-1 (1), Leu 1.99(2), Val. 0.89 (1), Asu(OAl) 0.95 (1).

EXAMPLE 8

Deprotection of the α-amino of the terminal amino residuum (Ser) and ofthe side chain of the residuum Asu. ObtainingH-Ser(tBu)-Asn-Leu-Ser(tBu)-Thr(tBu)-Asu-Val-Leu-Gly-HMPB-Ile-pMBHA(SEQ. ID NO:3) orH-Ser(tBu)-Asn-Leu-Ser(tBu)-Thr-(tBu)-Asu-Val-Leu-Gly-Wang (SEQ. IDNO:3) resin

Once the synthesis of the totally deprotected peptide is complete, therefollows the process of deprotecting the -amino group of the terminalresiduum (protected with Fmoc). This deprotection is carried out bytreating the resin, washed in DHF, with a 20% solution of piperidine/DMFtwice for 1 and 5 minutes. The resin is washed with DMF, DCM and isdried at low pressure.

Afterwards the side chain of the Asu residuum is deprotected. 200 mg ofthe peptide-resin are into a reactor provided with a filter plate in anatmosphere of helium. A separate solution is also prepared in anatmosphere of helium, said solution containing 7 mg of Pd(PPh₂)₄, 250 mgof PPh and 200 μl of morpholine, all dissolved in 3 ml of THF which haspreviously degassed by bubbling with helium. This solution is added tothe peptide-resin and the mixture is kept under helium for 60 hours,shaking periodically. When this time has elapsed the peptide-resin isfiltered and washed thoroughly with THF, DMF and DCM. Finally it isdried. In order to evaluate the degree of deprotection of the allylgroup, 20 mg of the peptide-resin are treated with 950 μl of TFA, 30 μlof DCM and 20 μl of anisole for 2 hours at room temperature in a reactorprovided with a filter plate. The filtrate is collected in a tube withcold, dry diethyl ether. The precipitation of the free peptide isobserved and after centrifuging the floating material is decanted. Thepellet is resuspended once again in cold, dry ether. This operation isrepeated five times. Afterwards the pellet is dried and then dissolvedin 1 ml of 10% acetic acid solution. 40 μl of the peptide solution areinjected into HPLC with a gradient of 5-85%, B, where A: H₂ O 0.045% TFAand: B: CH₃ CN 0.035% TFA, in a Vydac column C₁₈ 5 μm, 25×0.46 cm. Thedeprotection varies around 90%.

EXAMPLE 9

Cycling of 1-9 peptide on resin. Obtaining ##STR21##

The fragment 1-9 is cycled with the terminal and acid amine groups ofthe side chain of the Asu residuum. This is also carried out in reactorwith a filter plate. 60 mg of the peptide-resin, 16.2 μl of DIPCDI (6equivalents) and 15.6 mg of HOBt (6 equivalents) are introduceddissolved in DMF. The reaction is checked using the Kaiser test andafter 21 hours the resin is filtered and washed with DMF and DCM. Inorder to evaluate the purity of the peptide the same process asdescribed for the deprotected peptide in example 8 is carried out.

EXAMPLE 10

Liberation of cyclic 1-9 fragment from resin. Obtaining ##STR22##

The peptide-resinH-Ser(tBu)-Asn-Leu-Ser(tBu)-Thr(tBu)-Asu-Val-Leu-Gly-Rinikerhandle-Ile-pMBHA is treated with a 1% solution of TFA in DCM 4 or 5times over intervals of 15 minutes. Pyridine is added to the filtratesuntil it they are neutralized and the DCM is eliminated at low pressure.The white solid obtained is washed repeatedly with water and then dried.

Alternatively, the peptide-resinH-Ser(tBu)-Asn-Leu-Ser(tBu)-Thr(tBu)-Asu-Val-Leu-Gly-Wang resin (SEQ. IDNO:4) is treated with a solution containing TFA/DCM/anisole in theproportions 95/3/2 for 2 hours in a reactor provided with a filterplate. When this time has elapsed the acidic solution is poured ontocold, dry ether and the solid which appears is separated bycentrifuging. This solid is washed with ether two more times and thendried.

EXAMPLE 11

Liberation of the totally or partially protected nonapeptide from theresin. Obtaining

FmocSer(tBu)-Asn-Leu-Ser(tBu)-Thr(tBu)-Asu(OAl)-Val-Leu-Gly--OH orFmocSer-Asn-Leu-Ser-Thr-Asu(OAl)-Val-Leu-Gly--OH (SEQ. ID NO:3).

The same procedure as the previous example is carried out for bothresins.

EXAMPLE 12

Incorporation of the Fmoc AM handle onto Boc-Ile-pMBHa. Obtaining p-(R,S)-a- 1-(9H-fluoren-9-II)metoxi-formamido!-2,4-dimetoxylbenzyll!-fenoxiacetamido-Ile-pMBHA

There then follows the incorporation of p- (R,S)-a- 1-(9H-fluoren-9-yl)methoxy-formamide!-2,4-dimethoxy benzyl!-phenoxy acetic acid (Fmoc-AM).This is carried out by reacting 4 g of the resin (0.69 mmoles/g ofresin), after it has been previously functionalized with the internalstandard (following the protocol described in example 1), with 2.23 g(4.14 mmoles, 1.5 equivalents) of p- (R,S)-a- 1-(9H-fluoren-9-yl)methoxy-formamide!-2,4-dimethoxy benzyl!-phenoxy acetic acid, 0.62 g(4.14 mmoles, 1.5 equivalents) of HOBt and 641 μl (4.14 mmoles, 1.5equivalents) using DHF as the solvent. The reaction time is 90'. Afterthis time has elapsed, the resin is washed five times with DCM and theKaiser test is used to check that there are no free amines. If thereare, the coupling process must be repeated.

EXAMPLE 13

Incorporation of the Fmoc-PAL handle onto Boc-Ile-pMBHa. Obtaining1-(9H-fluoren-9-II)metoxi-formamido!metll-3,5-dimetoxlvaleriamido-Ile-pMBHA

There then follows the incorporation of 1-(9H-fluoren-9-yl)methoxy-formamide! methyl-3,5-dimethoxyvaletic acid (Fmoc-PAL); This iscarried out by reacting 4 g of the resin (0.69 mmol/g of resin), afterit has been previously functionalized with the internal standard(following the protocol described in example 1), with 1.89 g (4.14 mmol,1.5 equivalents) of 1-(9H-fluoren-9-yl) methoxy-formamide!methyl-3,5-dimethoxyvaleric acid, 0.62 g (4.14 mmol, 1.5 equivalents) ofHOBt and 641 μl (4.14mmol, 1.5 equivalents) using DMF as the solvent.The reaction time is 90'. After this time has elapsed, the resin iswashed five times with DCM and the Kaiser is used to check that thereare no free amines. If there are, the coupling process must be repeated.

EXAMPLE 14

Incorporation of the remaining amino acids. Obtaining FmocLys(Boc)-Leu-Ser(tBu)-Gln-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pmc)-Thr(tBu)-AA'₂₅-AA'₂₆ -Gly-AA'₂₈ -Gly-Thr(tBu)-Pro-AM-Ile-pMBHA (SEQ. ID NO:1).

The incorporation or the remaining amino acids is carried out byfollowing a synthesis programme such as the one described below:

    ______________________________________                                        Step      Reagent      Repetitions                                                                             Time                                         ______________________________________                                        1         DMF          5         1'                                           2         pip/DMF 20%  1         1'                                           3         pip/DMF 20%  1         5'                                           4         DMF          5         1'                                           5         Fmoc aa      --        +                                            6         HOBt         --        +                                            7         DIPCDI       --        40'                                          8         DMF          5         1'                                           ______________________________________                                    

check with ninhydrin, if + return to 5, if - continue with step 1 andthe next amino acid.

In order to evaluate the synthetic purity of the totally deprotected10-31 peptide, 20 mg of FmocLys(Boc)-Leu-Ser(tBu)-Gln-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pmc)-Thr(tBu)-AA'₂₅ -AA'₂₆-Gly-AA'₂₈ -Gly-Thr (tBu)-Pro-AM-Ile-pMBHA (SEQ. ID NO:8) are treatedwith 900 μl of TFA, 50 μl of thioanisole, 30 μl of EDT and 20 μl ofanisole for 2 hours at room temperature in a reactor provided with afilter plate. The filtrate is collected in a tube with cold, dry diethylether. The precipitation of the free peptide is observed and aftercentrifuging the floating material is decanted. The pellet isresuspended once again in cold, dry ether to eliminate the scavangers(EDT, thloanisole, anisole). This operation is repeated five times.Afterwards the pellet is dried and then dissolved in 1 ml of a solutionof 10% acetic acid. 40 μl of the peptide solution are injected into HPLCwith a gradient of 5-85% B, where A: H₂ O 0.045% TFA and B: CH₃ CN 0.035% TFA, Vydac C 5 μm, 25×0.46 cm. An amino acid analysis of a hydrolysisof the peptide-resin at 150° C. for 3 hours with a mixture ofHCl/propanoic acid gives the following composition Asp 1.06 (1), Thr 2.8or 4.0 (3 or 4), Ser 0.96 or 2.0 (1 or 2), Glu 3.01 (3), Gly 2.2 (2),Pro 1.98 (2), Ile 0.9 (1), Leu 3.0 (3), Tyr 0.8 (1), His 0.92 (1), Lys1.8 (2), Arg 1.03 (1), Ala 1.1 or 0 (1 or 0), Val 0.97 or 0 (1 or 0).

EXAMPLE 15

Incorporation of the protected or deprotected and cycled nonapeptideonto the peptide-resin of the protected 10-31 fragment 1. Obtaining##STR23##

2.03 g of the peptide-resin 10-31 are treated with piperidine/DMF for 3minutes. The operation is repeated two more times and the resin is thenwashed 5 times for 1 minute with DMF. 315 mg (2.5 equivalents) of HBTU(or alternatively the mmoles equivalents of PyBOP, 431 mg) and 124 mg(2.5 equivalents) of HOBT dissolved in DMF are added to the resin,forming the most homogeneous mass possible with the resin. 2.5equivalents of totally protected 1-9 cyclic peptide 1-9 (922 mg) ortotally deprotected cyclic peptide 1-9 (741 mg) are dissolved in theminimum quantity possible of DMF and added to the resin. Finally 296 μl(5 equivalents) of DIEA are added. The resin is shaken well until it ishomogeneous. The reaction acquires an orange colour. One hour and 30minutes later the Kaiser test on one aliquot part of the resin gives anegative result and the incorporation reaction can be consideredcomplete. The resin is filtered and washed repeatedly with DMF.

EXAMPLE 16

Linear synthesis of fragment 10. ObtainingFmocSer(tBu)-Asn-Leu-Ser(tBu)-Thr(tBu)-Asu(OAl)-Val-Leu-Gly-Lys(Boc)-Leu-Ser(tBu)-Gln-Glu(OtBu)-Leu-His(Trt-Lys(Boc)-Leu-Gln-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pmc)-Thr(tBu)-AA'₂₅-AA'₂₆ -Gly-AA'₂₆ -Gly-Thr(tBu)-Pro-AM-Ile-pMBHA (SEQ. ID NO:2)

There then follows the incorporation of all of the amino acids onto thepeptide-resin obtained as explained in examples 12 and 13, i.e. ontoAM-Ile-pMBHA or PAL-Ile-pMBHA. This incorporation is carried out in thesame way as indicated in example 14, as is the evaluation of thesynthetic purity of the final product. An amino acid analysis of ahydrolysis of the peptide-resin at 150° C. for 3 hours with a mixture ofHCl/propanoic acid gives the following composition Asp 2.08 (2), Thr3.76 or 4.2 (4 or 5), Ser 2.84 or 3.62 (3 or 4), Glu 3.01 (3), Gly 3.3(3), Pro 1.98 (2), Ile 0.9 (1), Leu 4.95 (5), Tyr 0.8 (1), His 0.92 (1),Lys 1.8 (2), Arg 1.03 (1), Ala 1.1 or 0 (1 or 0), Val 1.95 or 0.9 (2 or1), Asu(OAl) 0.96 (1).

EXAMPLE 17

Incorporation of the protected or deprotected nonapeptide withoutcycling onto the peptide-resin of the protected 10-31 fragment 1.Obtaining:FmocSer(tBu)-Asn-Leu-Ser(tBu)-Thr(tBu)-Asu(OAl)-Val-Leu-Gly-Lys(Boc)-Leu-Ser(tBu)-Gln-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln-Thr(tBu)-Tyr(tBu)-Pro-Art(Pmc)-Thr(tBu)-AA'₂₅ -AA'₂₆ -Gly-AA'₂₈₉ -Gly-Thr(tBu)-Pro-AM-Ile-pMBHAor FmocSer-Asn-Leu-Ser-Thr-Asu-(OAl)Val-Leu-Gly-Lys(Boc)-Leu-Ser(tBu)-Gln-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln-Thr(tBu)-Tyr(tBu)-Pro-Art(Pmc)-Thr(tBu)-AA'₂₅-AA'₂₆ -Gly-AA'₂₆ -Gly-Thr (tBu)-Pro-Am-Ile-pMBHA (SEQ. ID NO:2)

Follow the same method as described in the previous example.

EXAMPLE 18

Deprotection of the terminal O-amino and the side chain of the residuumAsu followed by cycling of the peptide 1-31. Obtaining: ##STR24##

The same procedure as described in example 8 is carried out for thedeprotection and that described in example 9 for cycling.

EXAMPLE 19

Breaking the resin and deprotection of the peptide 1-31 in any of itsforms. Obtaining: ##STR25##

The dry peptide-resin 1-31 is treated with TFA/DCM/anisole (95:3:2) for2 hours at room temperature. It is then poured onto 100 ml of cold, drydiethyl ether. The white precipitate which is obtained is separated bycentrifuging. The solid is resuspended once again in diethyl ether andcentrifuged again. This operation is repeated five more times. Finally,the solid obtained is dried and then, dissolved in 10% AcOH, is purifiedwith an HPLC preparation with a gradient of 5-65% B, where Al H₂ O 0.05%TFA and B: CH₃ CN 0.035% TFA, Vydac C18 15-20 μm, 25×1 cm.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 8                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:22 amino acids                                                     (B) TYPE:amino acid                                                           (C) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:peptide                                                    (v) FRAGMENT TYPE:C-Terminal type                                             (ix) FEATURE:                                                                 (D) OTHER INFORMATION:                                                        Lys at position 1 substituted with                                            t- butoxycarbonyl                                                             Ser at position 3 substituted with                                            tert- butyl                                                                   Glu at position 5 substituted with                                            tert- butyl                                                                   His at position 7 substituted with trityl                                     Lys at position 8 substituted with                                            t- butoxycarbonyl                                                             Thr at position 11 substituted with                                           tert- butyl                                                                   Tyr at position 12 substituted with                                           tert- butyl                                                                   Arg at position 14 substituted with                                           2,2,5,7,8- pentamethylchroman-6-sulphonyl                                     Thr at position 15 substituted with                                           tert- butyl                                                                   Xaa at position 16 being Asn or Asp                                           substituted with tert-butyl                                                   Xaa at position 17 being Val or Thr                                           substituted with tert-butyl                                                   Xaa at position 19 being Ala or Ser                                           substituted tert-butyl                                                        Thr at position 21 substituted with                                           tert- butyl                                                                   Pro at position 22 is attached to a                                           polymer bead.                                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       LysLeuSerGlnGluLeuHisLysLeuGlnThrTyrProArgThrXaa                              151015                                                                        XaaGlyXaaGlyThrPro                                                            20                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:31 amino acids                                                     (B) TYPE:amino acid                                                           (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:peptide                                                    (ix) FEATURE:                                                                 (D) OTHER INFORMATION: (Corresponds to the amino                              acid sequence of Fragments 9 and 10):                                         Ser at position 1 substituted with                                            9- fluorenylmethoxy carbonyl and                                              substituted with tert-butyl (Fragment 10)                                     Ser at position 4 substituted with tert-butyl                                 Thr at position 5 substituted with tert-butyl                                 Xaa at position 6 being L-`-aminosuberic acid                                 substituted with O-allyl                                                      Lys at position 10 substituted with t-butoxy carbonyl                         Ser at position 12 substituted with tert-butyl                                Glu at position 14 substituted with tert-butyl                                His at position 16 substituted with trityl                                    Lys at position 17 substituted with t-butoxy carbonyl                         Thr at position 20 substituted with tert-butyl                                Tyr at position 21 substituted with tert-butyl                                Arg at position 23 substituted with 2,2,5,7,8                                 pentamethychroman-6-sulphonyl                                                 Thr at position 24 substituted with tert-butyl                                Xaa at position 25 is Asn or Asp substituted                                  with tert- butyl                                                              Xaa at position 26 is Val or Thr substituted                                  with tert- butyl                                                              Xaa at position 28 is Ala or Ser protected                                    by tert- butyl                                                                Thr at position 30 is substituted with tert-butyl                             Pro at position 31 is attached to the solid support                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      SerAsnLeuSerThrXaaValLeuGlyLysLeuSerGlnGluLeu                                 151015                                                                        HisLysLeuGlnThrTyrProArgThrXaaXaaGlyXaaGlyThrPro                              202530                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:9 amino acids                                                      (B) TYPE:amino acid                                                           (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:peptide                                                    (ix) FEATURE:C-Terminal                                                       (D) OTHER INFORMATION: (Corresponds to amino                                  acid sequence in Fragments 4 and 5)                                           Ser at position 1 is derivatized with                                         9- fluorenylmethoxycarbonyl (Fragments 4 and 5)                               and tert- butyl (Fragment 5)                                                  Ser at position 4 is derivatized with tert-butyl                              Thr at position 5 is derivatized with tert-butyl                              Xaa at position 6 being L-`-aminosuberic acid                                 is derivatized with O-allyl                                                   Gly at position 9 is attached to a Wang-type                                  polymer bead                                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       SerAsnLeuSerThrXaaValLeuGly                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:9 amino acids                                                      (B) TYPE:amino acid                                                           (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:peptide                                                    (ix) FEATURE:C-Terminal                                                       (D) OTHER INFORMATION: (Corresponds to amino                                  acid sequence in Fragments 2 and 3)                                           Ser at position 1 is cyclized with                                            the omega carboxcylic acid of aminosuberic                                    acid of position 6                                                            Ser at position 4 is derivatized with tert-butyl                              Thr at position 5 is derivatized with tert-butyl                              Xaa at position 6 being L-`-aminosurebic acid                                 Gly at position 9 is attached to a Wang-type                                  polymer bead                                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       SerAsnLeuSerThrXaaValLeuGly                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:31 amino acids                                                     (B) TYPE:amino acid                                                           (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:peptide                                                    (ix) FEATURE:                                                                 (D) OTHER INFORMATION: (Corresponds to the amino                              acid sequence of Fragments 6 and 7):                                          Ser at position 1 is cyclized with the                                        omega carboxcylic acid of aminosuberic                                        acid of position 6 and substituted with                                       tert-butyl (Fragment 7)                                                       Ser at position 4 substituted with tert-butyl                                 Thr at position 5 substituted with tert-butyl                                 Xaa at position 6 being L-`-aminosuberic acid                                 Lys at position 10 substituted with t-butoxy carbonyl                         Ser at position 12 substituted with tert-butyl                                Glu at position 14 substituted with tert-butyl                                His at position 16 substituted with trityl                                    Lys at position 17 substituted with t-butoxy carbonyl                         Thr at position 20 substituted with tert-butyl                                Tyr at position 21 substituted with tert-butyl                                Arg at position 23 substituted with 2,2,5,7,8                                 pentamethychroman-6-sulphonyl                                                 Thr at position 24 substituted with tert-butyl                                Xaa at position 25 is Asn or Asp substituted                                  with tert- butyl                                                              Xaa at position 26 is Val or Thr substituted                                  with tert- butyl                                                              Xaa at position 28 is Ala or Ser protected                                    by tert- butyl                                                                Thr at position 30 is substituted with tert-butyl                             Pro at position 31 is attached to the solid support                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                      SerAsnLeuSerThrXaaValLeuGlyLysLeuSerGlnGluLeu                                 151015                                                                        HisLysLeuGlnThrTyrProArgThrXaaXaaGlyXaaGlyThrPro                              202530                                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:31 amino acids                                                     (B) TYPE:amino acid                                                           (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:peptide                                                    (ix) FEATURE:                                                                 (D) OTHER INFORMATION: (Corresponds to the amino                              acid sequence of carbocalcitonin):                                            Ser at position 1 is cyclized with the                                        omega carboxcylic acid of aminosuberic                                        acid of position 6                                                            Xaa at position 6 being L-`-aminosuberic acid                                 Xaa at position 25 is Asn or Asp                                              Xaa at position 26 is Val or Thr                                              Xaa at position 28 is Ala or Ser                                              Pro at position 31 is carboxylamide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                      SerAsnLeuSerThrXaaValLeuGlyLysLeuSerGlnGluLeu                                 151015                                                                        HisLysLeuGlnThrTyrProArgThrXaaXaaGlyXaaGlyThrPro                              202530                                                                        (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:10 amino acids                                                     (B) TYPE:amino acid                                                           (D) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:peptide                                                    (ix) FEATURE:C-Terminal                                                       (D) OTHER INFORMATION:                                                        Ser at position 1 is derivatized with                                         9- fluorenylmethoxycarbonyl tert-butyl                                        Ser at position 4 is derivatized with tert-butyl                              Thr at position 5 is derivatized with tert-butyl                              Xaa at position 6 being L-`-aminosuberic acid                                 is derivatized with O-allyl                                                   Gly at position 9 is substituted with Riniker handle                          Ile at position 10 is derivatized with pera-methyl                            benzhydrilamine                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       SerAsnLeuSerThrXaaValLeuGlyIle                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:23 amino acids                                                     (B) TYPE:amino acid                                                           (C) TOPOLOGY:linear                                                           (ii) MOLECULE TYPE:peptide                                                    (v) FRAGMENT TYPE:C-Terminal type                                             (ix) FEATURE:                                                                 (D) OTHER INFORMATION:                                                        Lys at position 1 substituted with                                            t- butoxycarbonyl                                                             Ser at position 3 substituted with                                            tert- butyl                                                                   Glu at position 5 substituted with                                            tert- butyl                                                                   His at position 7 substituted with trityl                                     Lys at position 8 substituted with                                            t- butoxycarbonyl                                                             Thr at position 11 substituted with                                           tert- butyl                                                                   Tyr at position 12 substituted with                                           tert- butyl                                                                   Arg at position 14 substituted with                                           2,2,5,7,8- pentamethylchroman-6-sulphonyl                                     Thr at position 15 substituted with                                           tert- butyl                                                                   Xaa at position 16 being Asn or Asp                                           substituted with tert-butyl                                                   Xaa at position 17 being Val or Thr                                           substituted with tert-butyl                                                   Xaa at position 19 being Ala or Ser                                           substituted tert-butyl                                                        Thr at position 21 substituted with                                           tert- butyl                                                                   Pro at position 22 is substituted with                                        p- (R,S)- 2,4-dimethoxybenzyl!-phenoxyacetic acid                             Ile at position 23 is substituted with                                        para- methylbenzhydrilamine                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       LysLeuSerGlnGluLeuHisLysLeuGlnThrTyrProArgThrXaa                              151015                                                                        XaaGlyXaaGlyThrProIle                                                         20                                                                            __________________________________________________________________________

We claim:
 1. A procedure for obtaining carbocalcitonin and itspharmaceutically acceptable acid addition salts or complexes thereof, bymeans of solid phase synthesis on polymeric supports and with theintervention of Fmoc/tBu characterized in that it comprises the stagesof:(a) condensing fragment 1 (SEQ. ID NO.: 1) consisting of thedocosapeptide corresponding to the carboxamide end of thecarbocalcitonin sequence, protected and anchored on resin, Fragment 1having the sequence:Lys(Boc)-Leu-Ser(tBu)-Gln-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pmc)-Thr(tBu)-AA'₂₅-AA'₂₆ -Gly-AA'₂₆ -Gly-Thr-Pro-®(SEQ. ID NO: 1)wherein -® has thefollowing formula with bridge group, internal structure and reticulatepolymer: ##STR26## with fragment 2, consisting of the nonapeptidecorresponding to the amino end of the carbocalcitonin sequence, with thecycle formed and with the absence of the protective groups for the sidechains Ser and Thr, Fragment 2 having the sequence: ##STR27## or,alternatively, with fragment 3, also consisting of the nonapeptidecorresponding to the amino end, with the cycle formed, but with the sidechains of the residue Ser and Thr protected with the group tBu, Fragment3 having the sequence: ##STR28## b) subjecting the peptide-resinresulting from the condensation and cycle formation reactions, that is,fragments 6 or 7 (SEQ. ID NO.: 5) wherein AA'₂₅ is selected from thegroup consisting of Asp and Asn, AA'₂₆ is selected from the groupconsisting of Val and Thr, and AA'₂₈ is selected from the groupconsisting of Ala and Ser, fragments 6 (X═H) and 7 (X═tBu) having thesequence: ##STR29## to an acidic treatment of deprotection of the sidechains and deanchoring of the resin to obtain the carbocalcitoninpeptide.
 2. A procedure according to claim 1 characterized in thatfragments 2 and 3 (SEQ. ID NO.: 4) are obtained frompara-methylbenzhydrilamine resin by incorporating the internal standardand the bridge group between the resin and the sequence of amino acids,this being followed by linear synthesis, residuum by residuum, until the9 amino acids of fragments 2 or 3 have been incorporated.
 3. A procedureaccording to claim 2, wherein the linear synthesis of the fragment of 9amino acids, the group Fmoc is used for the amino end in all cases, andfor the side chains Lys, Ser, Thr, Cys and Asn the protective groupsindicated for said chains in the formula of fragments 2, 3 (SEQ. ID NO.:4), 4 (SEQ. ID NO.: 3) and 5 (SEQ. ID NO.: 4) are used.
 4. A procedurefor obtaining carbocalcitonin and its pharmaceutically acceptable acidaddition salts or complexes thereof, by means of solid phase synthesison polymeric supports and with the intervention of Fmoc/tBu comprisingthe steps of:(a) condensing fragment 1 (SEQ. ID NO.: 1) consisting ofthe docosapeptide corresponding to the carboxamide end of thecarbocalcitonin sequence, protected and anchored on resin; fragment 1having the sequence:Lys(Boc)-Leu-Ser(tBu)-Gln-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pmc)-Thr(tBu)-AA'₂₅ -AA'₂₆ -Gly-AA'₂₈ -Gly-Thr-Pro-®(SEQ. ID NO.: 1)wherein -® has the following formula with bridge group, internalstructure and reticulate polymer: ##STR30## with fragment 5 having thesequence:FmocSer(tBu)-Asn-Leu-Ser(tBu)-Thr(tBu)-Asu(OAl)-Val-Leu-Gly--OH (SEQ. IDNO.: 3)to obtain the peptide-resin corresponding to fragment 10 havingthe sequence:FmocSer(tBu)-Asn-Leu-Ser(tBu)-Thr(tBu)-Asu(OAl)-Val-Leu-Gly-Lys(Boc)-Leu-Ser(tBu)-Gln-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pmc)-Thr(tBu)-AA'₂₅-AA'₂₆ -Gly-AA'₂₈ -Gly-Thr-Pro-® (SEQ. ID NO.: 2)which is subjected to areaction of deprotection and cyclization to obtain fragment 7 (SEQ. IDNO.: 5) which is in turn subjected to the treatment of de-anchoring fromthe resin and then purified to obtain carbocalcitonin wherein AA'₂₅ isselected from the group consisting of Asp and Asn, AA'₂₆ is selectedfrom the group consisting of Val and Thr, and AA'₂₈ is selected from thegroup consisting of Ala and Ser.
 5. A procedure according to claims 1 or4, characterized in that fragment 1 (SEQ. ID NO.: 1) is obtained frompara-methylbenzhydrilamine resin by incorporating the internal standardselected from Ile or Ala and the bridge group selected from p(R,S)-2,4-dimethoxybenzyl) phenoxy acetic acid oraminomethyl-3,5-dimethoxyphenoxyvaleric acid between the resin and thesequence of amino acids, this being followed by linear synthesis,residuum by residuum, until the 22 amino acids have been incorporated.6. A procedure according to claim 5 characterized in that during thelinear synthesis of the fragment of 22 amino acids the group Fmoc isused for the amino end in all cases and for the side chains Lys, Ser,Thr, Tyr, Arg, Glu, His, and Asp the protective groups indicated forsaid chains of the formula for fragment 1 (SEQ. ID NO.: 1) are used. 7.A procedure according to claims 1, or 4 characterized in that fragments2, 3 (SEQ. ID NO.: 4), and 5 (SEQ. ID NO.: 3) are obtained frompara-methylbenzhydrilamine resin by incorporating the internal standardand the bridge group between the resin and the sequence of amino acids,this being followed by linear synthesis, residuum by residuum, until the9 amino acids have been incorporated.
 8. A procedure according to claim7, characterized in that in the linear synthesis of the fragment of 9amino acids, the group Fmoc is used for the amino end in all cases, andfor the side chains Lys, Ser, Thr, Cys and Asn the protective groupsindicated for said chains in the formula of fragments 2, 3 (SEQ. ID NO.:4) and 5 (SEQ. ID NO.: 4) are used.
 9. A procedure for obtainingcarbocalcitonin and its pharmaceutically acceptable acid addition saltsor complexes thereof, by means of solid phase synthesis on polymericsupports and with the intervention of Fmoc/tBu comprising the stepsof:(a) condensing fragment 1 (SEQ. ID NO.: 1) consisting of thedocosapeptide corresponding to the carboxamide end of thecarbocalcitonin sequence, protected and anchored on resin; fragment 1having the sequence:Lys(Boc)-Leu-Ser(tBu)-Gln-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pmc)-Thr(tBu)-AA'₂₅-AA'₂₆ -Gly-AA'₂₈ -Gly-Thr-Pro-®(SEQ. ID NO.: 1)wherein -® ha thefollowing formula with bridge group, internal structure and reticulatepolymer: ##STR31## with fragment 4 having the sequence:FmocSer-Asn-Leu-Ser-(tBu)-Thr-(tBu)-Asu(OAl)-Val-Leu-Gly--OH (SEQ. IDNO.: 3)to obtain the peptide-resin which is fragment 9 having thesequence:FmocSer-Asn-Leu-Ser(tBu)-Thr(tBu)-Asu-(OAl)-Val-Leu-Gly-Lys(Box)-Leu-Ser(tBu)-Gln-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pmc)-Thr(tBu)-AA'₂₅ -AA'₂₆ -Gly-AA'₂₈ -Gly-Thr-Pro-® (SEQ. IDNO.:2)which is subjected to the reaction steps of deprotection,cyclization, and de-anchoring from the resin and then purified to obtaincarbocalcitonin wherein AA'₂₅ is selected from the group consisting ofAsp and Asn, AA'₂₆ is selected from the group consisting of Val and Thr,and AA'₂₈ is selected from the group consisting of Ala and Ser. the sidechains Lys, Ser, Thr, Tyr, Arg, Glu, His, Gln and Asp the protectivegroups are as indicated for said chains in the formula of fragment 1(SEQ. ID NO.: 1).
 10. A procedure according to claim 9 characterized inthat fragment 1 (SEQ. ID NO.: 1) is obtained frompara-methylbenzhydrilamine resin by incorporating the internal standardselected from Ile or Ala and the bridge group selected from p(R,S)-2,4-dimethoxybenzyl)phenoxy acetic acid oraminomethyl-3,5-dimethoxypherioxyvaleric acid between the resin and thesequence of amino acids, this being followed by linear synthesis,residuum by residuum, until the 22 amino acids have been incorporated.11. A procedure according to claim 10 characterized in that during thelinear synthesis of the fragment of 22 amino acids the group Fmoc isused for the amino end in all cases, and for the side chains Lys, Ser,Thr, Tyr, Arg, Glu, His, Gln, and Asp the protective groups are asindicated for said chains in the formula of fragment 1 (SEQ. ID NO.: 1).12. A method for forming carbocalcitonins in which a cyclic structure isformed between the side chain carboxylic acid of Asu and an aminofunctionality of a different amino acid comprising the steps of:(a)incorporating Fmoc-Asu(ω-allyl)--OH into a polypeptide; (b)incorporating at least one additional amino acid into the polypeptide ofstep (a); (c) removing the ω-allyl group from the carboxylic acid sidechain of Asu; and (d) cyclizing the polypeptide wherein the cyclicstructure is formed between the side chain carboxylic acid of Asu and anamino functionality of a different amino acid, to form carbocalcitonin.13. The method of claim 12 wherein the carbocalcitonins are selectedfrom the group consisting of salmon, eel, human, porcine and bovinecarbocalcitonins.
 14. The method of claim 12 further comprising thesteps of forming fragments 1 (SEQ. ID NO.: 1), 2 (SEQ. ID NO.: 4) and 3(SEQ. ID NO.: 4) and then coupling fragment 1 (SEQ. ID NO.: 1) with oneof fragments 2 (SEQ. ID NO.: 4) or 3 (SEQ. ID NO.: 4) to form one offragments 6 (SEQ. ID NO.: 5) or 7 (SEQ. ID NO.: 5) wherein AA'₂₅ isselected from the group consisting of Asp and Asn, AA'₂₆ is selectedfrom the group consisting of Val and Thr, and AA'₂₈ is selected from thegroup consisting of Ala and Ser, fragment 6(X=H) and 7(X=tBu) having thesequence: ##STR32## wherein -® has the following formula with bridgegroup, internal structure and reticulate polymer: ##STR33##
 15. Themethod of claim 12 further comprising the steps of forming fragments 1(SEQ. ID NO.: 1) and 4 (SEQ. ID NO.: 4) and then coupling fragment 1(SEQ. ID NO.: 1) with fragment 4 (SEQ. ID NO.: 4) to form fragment 9(SEQ. ID NO.: 2) wherein AA'₂₅ is selected from the group consisting ofAsp and Asn, AA'₂₆ is selected from the group consisting of Val and Thr,and AA'₂₈ is selected from the group consisting of Ala and Ser, fragment9 having thesequence:FmocSer-Asn-Leu-Ser-Thr-Asu-(OAl)-Val-Leu-Gly-Lys(Boc)-Leu-Ser(tBu)-Gln-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pmc)-Thr(tBu)-AA'₂₅-AA'₂₆ Gly-AA'₂₈ -Gly-Thr-Pro-®(SEQ. ID NO. :2) wherein -® has thefollowing formula with bridge group, internal structure and reticulatepolymer: ##STR34##
 16. The method of claim 12 further comprising thesteps of forming fragments 1 (SEQ. ID NO.: 1) and 5 (SEQ. ID NO.: 3) andthen coupling fragment 1 (SEQ. ID NO.: 1) with fragment 5 (SEQ. ID NO.:3) to form fragment 10 (SEQ. ID NO.: 2) wherein in fragment 10 AA'₂₅ isselected from the group consisting of Asp and Asn, AA'₂₆ is selectedfrom the group consisting of Val and Thr, and AA'₂₈ is selected from thegroup consisting of Ala and Ser, fragment 10 having thesequence:FmocSer(tBu)-Asn-Leu-Ser(tBu)-Thr(tBu)-Asu(OAl)-Val-Leu-Gly-Lys(Boc)-Leu-Ser(tBu)-Gln-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pmc)-Thr(tBu)-AA'₂₅ -AA'₂₆ -Gly-AA'₂₈ -Gly-Thr-Pro-®(SEQ. ID NO.: 2)wherein -200 has the following formula with bridge group, internalstructure and reticulate polymer: ##STR35##
 17. A method for thesolid-phase synthesis of carbocalcitonin comprising the stepsof:selecting an insoluble solid support; chemically protecting thealpha-amino group of the carboxyl-terminal amino acid with Fmoc;coupling said carboxyl-terminal amino acid to said solid support;deprotecting said carboxyl-terminal amino acid; selecting andintroducing additional Fmoc-protected amino acids; selectingFmoc-Asu(ω-allyl)--OH as a protected Asu amino acid; and alternating thedeblocking and coupling reaction to form carbocalcitonin.
 18. A newpeptide fragment for use in the synthesis of carbocalcitonin having thefollowing sequence and structure, protected and anchored onresin:Lys(Boc)-Leu-Ser(tBu)-Gln-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pmc)-Thr(tBu)-Asp(OtBu)-Val-Gly-Ala-Gly-Thr-Pro-®(SEQ. ID NO.: 1)wherein -® has the following formula with bridge group, internalstructure and reticulate polymer: ##STR36##
 19. A new peptide fragmentfor use in the synthesis of salmon or eel carbocalcitonin having thefollowing sequence and structure: ##STR37##
 20. A new peptide fragmentfor use in the synthesis of salmon or eel carbocalcitonin having thefollowing sequence and protected structure: ##STR38##
 21. A new peptidefragment for use in the synthesis of salmon or eel carbocalcitoninhaving the following sequence and structure, protected and anchored onresin, wherein AA'₂₅ is selected from the group consisting of Asp andAsn, AA'₂₆ is selected from the groups consisting of Val and Thr, andAA'₂₈ is selected from the group consisting of Ala and Ser: ##STR39##wherein X is H and -® has the following formula with bridge group,internal structureand reticulate polymer: ##STR40##
 22. A new peptidefragment for use in the synthesis of salmon or eel carbocalcitoninhaving the following sequence and structure, protected and anchored onresin, wherein AA'₂₅ is selected from the group consisting of Asp andAsn, AA'₂₆ is selected from the group consisting of Val and Thr, andAA'₂₈ is selected from the group consisting of Ala and Ser: ##STR41##wherein -® has the following formula with bridge group, internalstructure and reticulate polymer: ##STR42##
 23. A new peptide fragmentfor use in the synthesis of salmon or eel carbocalcitonin having thefollowing partially protected sequence andstructure:FmocSer-Asn-Leu-Ser-(tBu)-Thr-(tBu)-Asu(OAl)-Val-Leu-Gly--OH(SEQ. ID NO.: 3).
 24. A new peptide fragment for use in the synthesis ofsalmon or eel carbocalcitonin having the following sequence andstructure, protected and anchored in resin, wherein AA'₂₅ is selectedfrom the group consisting of Asp and Asn, AA'₂₆ is selected from thegroup consisting of Val and Thr, and AA'₂₈ is selected from the groupconsisting of Ala and Serhaving:FmocSer-Asn-Leu-Ser-Thr-Asu(OAl)-Val-Leu-Gly-Lys(Boc)-Leu-Ser(tBu)-Gln-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pmc)-Thr(tBu)-AA'₂₅-AA'₂₆ -Gly-AA'₂₈ -Gly-Thr-Pro-®(SEQ. ID NO.: 2) wherein -® has thefollowing formula with bridge group, internal structure and reticulatepolymer: ##STR43##
 25. A new peptide fragment for use in the synthesisof salmon or eel carbocalcitonin having the following protected sequenceandstructure:FmocSer(tBu)-Asn-Leu-Ser(tBu)-Thr(tBu)-Asu(OAl)-Val-Leu-Gly--OH(SEQ. ID NO.: 3).
 26. A new peptide fragment for use in the synthesis ofsalmon or eel carbocalcitonin having the following sequence andstructure, protected and anchored on resin, wherein AA'₂₅ is selectedfrom the group consisting of Asp and Asn, AA'₂₆ is selected from thegroup consisting of Val and Thr, and AA'₂₈ is selected from the groupconsisting of Ala andSer:FmocSer(tBu)-Asn-Leu-Ser(tBu)-Thr(tBu)-Asu(OAl)-Val-Leu-Gly-Lys(Boc)-Leu-Ser(tBu)-Gln-Glu(OtBu)-Leu-His(Trt)-Lys(Boc)-Leu-Gln-Thr(tBu)-Tyr(tBu)-Pro-Arg(Pmc)-Thr(tBu)-AA'₂₅-AA'₂₆ -Gly-AA'₂₈ -Gly-Thr-Pro-®(SEQ. ID NO. 2) wherein -® has thefollowing formula with bridge group, internal structure and reticulatepolymer: ##STR44##